Double-stranded RNA dependent protein kinase (alternatively, "PKR") is a serine/threonine protein kinase which exerts antiviral and anticellular functions. It can be induced by interferon (Meurs, et al., Cell 62:379-390 (1990); Sen, et al. J. Biol. Chem. 267:5017-5020 (1992); Hovanessian, A. G., J. Interferon Res. 9:641-647 (1989)). PKR is involved in regulating a number of physiologic processes. These include cell growth and differentiation (Petryshyn, et al., Proc. Natl. Acad. Sci. USA 85:1427-1431 (1988); Petryshyn, et al., J. Biol. Chem. 259:14736-14742 (1984); Judware, et al., Mol. Cell. Biol. 11:3259-3267 (1991), tumor suppression (Koromilas, et al., Science 257:1685-1689 (1992); Meurs, et al., Proc. Natl. Acad. Sci. USA 90:232-236 (1993)), and modulation of signal transduction pathways (Leonardo, et al., Cell 57:287-294 (1989); Kumar, et al., Proc. Natl. Acad. Sci. U.S.A. 91:6288-6292 (1994); Maran, et al., Science 265:789-792 (1994)).
These cellular effects of PKR have generally been attributed to translational regulation (Farrell, et al., Cell 11:187-200 (1977); Petryshyn, et al., Methods Enzymol. 99:346-362 (1983); Samuel, C. E., Proc. Natl. Acad. Sci. U.S.A. 76:600-604 (1979)). In the presence of low concentrations of double-stranded RNA (dsRNA), divalent cations and ATP, PKR undergoes a phosphorylation which is required to convert the enzyme from a latent to an active protein kinase (Edery, et al., Cell 56:303-312 (1989); Lebleu, et al., Proc. Natl. Acad. Sci. USA 73:3107-3111 (1976); Petryshyn, et al., Methods Enzymol. 99:346-362 (1983)). Paradoxically, the phosphorylation and activation is prevented by high concentrations of dsRNA (Farrell, et al., Cell 11:187-200 (1977); Hunter, et al., J. Biol. Chem. 250:7887-7891 (1975)). Once activated, PKR phosphorylates the alpha subunit of the eukaryotic initiation factor 2 (elF-2 alpha) (Farrell, et al., Cell 11:187-200 (1977); Lebleu, et al., Proc. Nat. Acad. Sci. USA 73:3107-3111 (1976); Petryshyn, et al., Methods Enzymol. 99:346-362 (1983)), which in turn, results in inhibition of protein synthesis (London, et al. (Boyer, et al. (eds)), The Enzymes, vol. 18. Academic Press, New York (1987); Hershey, J. W., J. Biol. Chem. 264:20823-20826 (1989)). The antiviral effect of PKR is believed to be mediated the phosphorylation of elF-2 alpha. However, it is not known whether PKR's anticellular effect is due to phosphorylation of elF-2 alpha, 1 kappa B or another unknown substrate (Lee, et al. Virology 231:81-88 (1997)).
The mechanism by which PKR interacts with dsRNA is unclear. Neither the spatial, structural or sequence requirements within the RNA or the protein itself are sufficiently resolved to fully understand the dynamics of this interaction. Since a diverse group of viral RNAs interact and modulate the activity of PKR (Clarke, et al., Nucleic Acids Res. 19:243-248 (1991); Kitajewski, et al., Cell 45:195-200 (1986); Hovanessian, A. G., J. Interferon Res. 9:641-647 (1989); Hunter, et al., J. Biol. Chem. 250:7887-7891 (1975); SenGupta, et al. Nucleic Acids Res. 17:969-978 (1989); Roy, et al., J. Virol 65:632-640 (1991); Edery, et al., Cell 56:303-312 (1989); Judware, et al., J. Interferon Res. 13:153-160 (1993); Biscboff, et al., Virology 172:106-115 (1989)), there does not appear to be sequence specificity. However, there is a dependency on both the length of the double-strandedness and its secondary structure (Manche, et al., Mol. Cell. Biol. 12:5238-5248 (1992); Ghadge, et al., J. Virol. 68:4137-4151 (1994); Hunter, et al., J. Biol. Chem. 250:7887-7891 (1975); Edery, et al., Cell 56:303-312 (1989)). Tertiary structure is also likely to be important. Several viral RNAs inhibit the activation of PKR (Kitajewski, et al., Cell 45:195-200 (1986); Clarke, et al., Nucleic Acids Res. 19:243-248 (1991); Ghadge, et al., J. Virol. 68:4137-4151 (1994)), while others are efficient activators (Hovanessian, A. G., J. Interferon Res. 9:641-647 (1989)). The TAR sequence of HIV-1 mRNA transcript has been shown to both activate (Edery, et al., Cell 56:303-312 (1989); SenGupta, et al. Nucleic Acids Res. 17:969-978 (1989); Judware, et al., J. Interferon Res. 13:153-160 (1993)) and prevent activation (Gunnery, et al., Proc. Natl. Acad. Sci. USA 87:8687-8691 (1990)) of PKR at low concentrations.
Both human (Meurs, et al., Cell 62:379-390 (1990)) and murine PKR (Feng, et al., Proc. Natl. Acad. Sci. USA 89: 5447-5451 (1992); Baier, et al., Nucleic Acids Res. 21:4830-4835 (1993)) have been cloned and sequenced and these two cDNAs share extensive nucleotide sequence identity (Feng, et al., Proc. Natl. Acad. Sci. USA 89: 5447-5451 (1992)). Results from several studies have reported that the RNA-binding domain of PKR is localized to the N-terminal portion of the kinase. Feng, et al., Proc. Natl. Acad. Sci. USA 89: 5447-5451 (1992); McCormack, et al., Virology 188:47-56 (1992); Patel, et al., J. Biol. Chem. 269:18593-18598 (1994); Green, et al., Genes Dev. 6:2478-2490 (1992); Patel, et al., J. Biol. Chem. 267:7671-7676 (1992). Although deletions of several short portions of PKR sequence rich in positively charged residues have been shown to diminish dsRNA-induced PRK activation, no discrete PKR region or amino acid sequence motif which is both necessary and sufficient to bind to regulatory dsRNA was known prior to this invention (Feng, et al., Proc. Natl. Acad. Sci. USA 89: 5447-5451 (1992)).
Thus, prior to this invention, the existence of a defined linear, non-conformationally dependent dsRNA-binding region of PKR, which is both necessary and sufficient to bind to dsRNA, was unknown.
Furthermore, PKR antagonists were unknown. As PKR is a regulator of cell quiescence and cell death, such antagonists would be valuable for treating diseases or conditions associated with premature or induced cell death, such as the T cell depletion due to HIV-1 infection.
Thus, there exists a great need for inhibitors of PKR. The present invention fulfills these and other needs.